The present application claims priority to Korean Application No. 2001-73283 filed Nov. 23, 2001, the disclosure of which is incorporated herein by reference in its entity.
1. Field of the Invention
The present invention relates to a sensor for use in sample analysis and its applications, and more particularly, to a surface plasmon resonance sensor and an imaging system based on the principle of the surface plasmon resonance sensor.
2. Description of the Related Art
Surface plasmon is a quantized oscillation of free electrons that propagates along the surface of a conductor such as a metal thin film. Surface plasmon is excited to cause resonance by an incident light beam entering a metal thin film through a dielectric medium such as a prism at an incident angle greater than a critical angle. This phenomenon is referred to as xe2x80x9csurface plasmon resonancexe2x80x9d (SPR). The incident angle of an incident light beam that causes resonance is very sensitive to changes in the refractive index of a material closest to the metal thin film. SPR sensors developed based upon the above principle have been widely used for quantification and qualification of a sample or measurement of a sample (thin film) thickness from changes in the refractive index of the sample displaced closest to the metal thin film.
FIG. 1 shows a typical SPR sensor based on the Kretschmann configuration. Referring to FIG. 1, the SPR includes a unit U composed of a dielectric medium 10 and a metal thin film 22 that induces SPR. A half-cylindrical or triangular prism made of transparent glass, such as BK7 and SF10, is often used for the dielectric medium 10. The metal thin film is formed of gold or silver with a thickness of 40-50 nm. The unit U is supported by a rotary plate 50 capable of rotating around a fixed shaft. A sample 23 of interest to be measured for changes in its refractive index within the surface plasmon field is brought into contact with the metal thin film 22 of the unit U.
In FIG. 1, reference numeral 30 denotes a light source fixed to emit an incident light beam 31 toward the metal thin film 22, and reference numeral 40 denotes a photodetector for measuring the intensity of the reflected light from the surface of the metal thin film 22. A monochromatic laser, a monochromic light emitting diode (LED), a white light source of multiple wavelengths, or a multiple-wavelength LED is often used as the light source 30.
SPR occurs when the wave vector of the incident light beam 31 parallel to the surface of the metal thin film 22 is equal to the wave vector of the surface plasmon wave. Thus, the following formula (1) is satisfied:                               n          ⁢                      xe2x80x83                    ⁢          sin          ⁢                      xe2x80x83                    ⁢                      θ            re                          =                                                            ϵ                1                            ⁢                              ϵ                2                                                                    ϵ                1                            +                              ϵ                2                                                                        (        1        )            
where n is the refractive index of the dielectric medium, xcex8re is the resonance angle, and ∈1 and ∈2 are the dielectric constants of the metal thin film 22 and the sample 23, respectively.
As is apparent from the formula (1) above, if the resonance angle xcex8re is given, the dielectric constant of the sample 23 can be calculated using the formula (1) and thus changes in the refractive index of the sample 23 or with respect to a reference sample can be observed. As a consequence, measurement of the thickness of the sample 23 if it is a thin film, or quantification and qualification of the sample adsorbed onto the metal thin film 22 can be implemented from the changes in the refractive index.
Resonance angle xcex8re can be measured using a variety of methods.
First, the fact that the intensity of the reflected light (or reflectance) 39 has a minimal value when the metal thin film 22 is excited to induce SPR by the incident light beam 31 is used. In this method, the intensity of the reflected light (or reflectance) 39 is measured while changing the incident angle xcex8 of the incident light beam 31, and the resonance angle xcex8re, the incident angle at which resonance occurs, is read from a plot of the intensity of the reflected light (or reflectance) 39 as a function of the incident angle xcex8. The intensity of the reflected light (or reflectance) 39 is measured while rotating the rotary plate 50 to vary the incident angle xcex8, in which a monochromic light source as the light source 30 and a prism with a constant refractive index as the dielectric medium 10 are used.
In a second method, a wavelength where SPR occurs is found by emitting the incident light beam 30 at a fixed incident angle xcex8 using a white light source of multiple wavelengths as the light source 30. As a result, the resonance angle xcex8re and resonance wavelength can be obtained simultaneously.
In a third method, the resonance angle xcex8re is measured by emitting a monochromic light from the light source 30 within the range of the incident angle to the center of the dielectric medium 10 and by receiving the light reflected from the surface of the metal thin film 22 with the same range of angles as the incident angle using a multi-channel photodetector, such as a photodiode array (PDA), as the photodetector 40. This method is disclosed in U.S. Pat. Nos. 4,889,427; 5,359,681; and 4,844,613.
The method of measuring the resonance angle xcex8re using a monochromatic light as in the first and third methods described above has about 10 times higher sensitivity than the second method using a white light source at a fixed incident angle. For this reason, the first and third methods have been used widely, and products based on the third method are available from Biocore and Texas Instrument.
FIG. 2 shows reflectances as a function of the incident angle of light measured using the SRP sensor of FIG. 1 for samples of different refractive indices. In FIG. 2, (1) is for water, (2) is for a sample with a refractive index difference of 10xe2x88x926 from water, and (3) is for a sample with a refractive index difference of 10xe2x88x923 from water.
An inset for a portion A in FIG. 2 shows changes in resonance angle with respect to changes in the refractive index of samples. A change in resonance angle (xcex94xcex8) by about 0.0001xc2x0 occurs between samples (1) and (2) having a refractive index difference of 10xe2x88x926. In measuring the resonance angle by the first and third methods described above, the rotary plate 50 used in the first method to vary the incident angle has an angular resolution limit of about 0.0001xc2x0 and the photodetector 40 such as a PDA which spatially splits the light reflected within a predetermined range of angles has a resolution limit of about 0.0001xc2x0. Thus, it is difficult for the SPR sensor with such a resolution limit to detect a minor change in refractive index less than 10xe2x88x926 or equivalent physical quantities, for example, protein adsorbed onto the surface of a metal thin film in an amount of less than several picograms per 1 mm2. In addition, adsorption of a material having a molecular weight less than 200 cannot be detected.
In the method of measuring reflectance at a fixed incident angle xcex1, a change in reflectance (xcex94R) for a refractive index difference of 10xe2x88x926 between samples is only 0.03% at xcex1=65.0304xc2x0. In consideration of the 0.2% resolution of a measuring system commonly used in the field, this method has a lower refractive index resolution than the methods for directly measuring the resonance angle.
To address the limitations of the SPR sensor, a coupled plasmon-waveguide resonance (CPWR) sensor, as shown in FIG. 3, has been developed. In FIG. 3, the same elements as those in FIG. 1 are denoted by the same reference numerals as those in FIG. 1.
Referring to FIG. 3, the CPWR sensor with improved sensitivity is a modification of the SPR sensor of FIG. 1. The CPWR sensor, which is disclosed in U.S. Pat. No. 5,991,488, includes a dielectric thin film 60 between the metal thin film 22 and the sample 23. The dielectric thin film 60 is formed as a single or multiple layers and acts as a waveguide. The dielectric thin film 60 is formed of a dielectric material, such as SiO2, Al2O3, TiO2, MgF2, and ZnS, to a thickness of 400-800 nm. Unlike the SPR sensor where surface plasmon waves propagate along the surface of the metal thin film 22, the incident light beam 31 is coupled into the surface plasmon mode between the surface of the metal thin film 22 and the dielectric thin film 60 deposited on the metal thin film 22 and propagates along the dielectric thin film 60. In the CPWR sensor having the configuration above, the CPWR or attenuated total reflection (ATR) leaky mode is observed at an angle smaller than the resonance angle of the SPR sensor.
FIG. 4 shows reflectances as a function of the incident angle of light measured using the CPWR sensor of FIG. 3 for samples of different refractive indices. In FIG. 4, (1) is for water, (2) is for a sample with a refractive index difference of 10xe2x88x926 from water, and (3) is for a sample with a refractive index difference of 10xe2x88x923 from water, as in FIG. 2.
As shown in FIG. 4, the CPWR sensor has a narrower range of resonance angle than the SPR sensor. Thus, the CPWR sensor is expected to be able to easily measuring changes in resonance angle for the samples of different refractive indices, compared to the SPR sensor. Actually, the CPWR sensor can measure the amount of protein adsorbed to a sample surface to a concentration of 0.5 pg/mm2 with 2-4 times improvement in resolution compared to the SPR sensor.
As shown in an inset for a portion A in FIG. 4, a change in resonance angle (xcex94xcex8) by about 0.00008xc2x0 occurs between samples (1) and (2) having a refractive index difference of 10xe2x88x926. Because the CPWR sensor of FIG. 3 also has an angular resolution limit of about 0.0001xc2x0, there is a need to improve the resolution of refractive index by using the rotary plate 50 or a multi-channel photodetector, such as a PDA, as the photodetector 40, which is capable of improve the angular resolution. However, technical problems hinder use of this approach. Moreover, aside from technical difficulties, due to the high cost involved, this approach is not economically feasible.
When reflectances are measured at a fixed incident angle xcex2 using the CPWR sensor of FIG. 4, a change in reflectance (xcex94R) for a refractive index difference of 10xe2x88x926 between samples is about 0.56% at xcex2=61.5665xc2x0, which is greater than the conventional SPR sensor. This result supports that the resolution of refractive index can be improved by increasing changes in reflectance with respect to incident angle variations, i.e., the slope of a curve of reflectance versus incident angle, by making the range of resonance angle narrow.
Theoretically, changes in reflectance with respect to incident angle variations can be increased by reducing the thickness of the dielectric thin film in the CPWR sensor. However, improving the resolution of refractive index by this method has limitations for the following reasons.
FIG. 5A shows reflectances as a function of the incident angle measured using CPWRs having different dielectric film thicknesses. In FIG. 5A, (a), (b), and (c) are for the cases where the dielectric thin film, for example, formed of TiO2, has a thickness of 138 nm, 135 nm, and 133 nm, respectively.
Referring to FIG. 5A, when the dielectric thin film is deposited to a thickness as small as 138 nm or less, the measurable range of reflectance becomes narrow, so it is difficult to select an appropriate incident angle. Finally, the width of resonance dip becomes partially broad at a dielectric film thickness of 133 nm. Therefore, improving the resolution of refractive index in the reflectance measurement method through the adjustment of dielectric film thickness is limited.
FIG. 5B shows absorbances as a function of the incident angle of light measured using CPWRs having different dielectric film thicknesses. In FIG. 5B, (a), (b), and (c) are for the cases where the dielectric thin film, for example, formed of TiO2, has a thickness of 138 nm, 135 nm, and 133 nm, respectively, as in FIG. 5A.
As shown in FIG. 5B, the pattern of the absorbance curve is maintained at a reduced dielectric film thickness of 133 nm with a steep slope portion as indicated by xe2x80x9cAxe2x80x9d. Therefore, a sensor with improved refractive index resolution can be implemented by measuring changes in absorbance, rather than reflectance, with respect to refractive index variations, at least within the range of the incident angle for the step slope portion xe2x80x9cAxe2x80x9d. However, the absorbance of the metal thin film cannot be measured using the CPWR sensor having the above-described structure.
To solve the above-described problems, it is an objective of the present invention to provide an active ion-doped waveguide-plasmon resonance (AID WPR) sensor with improved sensitivity over the conventional surface plasmon resonance (SPR) sensor and coupled plasmon-waveguide resonance (CPWR) sensor, and an imaging system based on the principle of the active ion-doped waveguide-plasmon resonance sensor.
To achieve the objective of the present invention, unlike the conventional SPR or CPWR sensor, which measures the resonance angle from the intensity of reflected light (reflectance) received by a photodetector with angular resolution, such as a photodiode array (PDA) or a photodetector which is supported by a rotary plate, a method of measuring the absorption of an incident light beam through surface plasmon resonance is used. The active ion-doped waveguide-plasmon resonance (AID WPR) according to the present invention is characterized in that it uses a dielectric thin film doped with active ions of an element or organic dye capable of fluorescing through absorption of an incident light beam, in proportional to the intensity of the absorbed light beam, and determines the absorption of the incident light beam from fluorescence variations of the active ions with improved refractive index resolution of samples.
In particular, the AID WPR sensor according to the present invention includes a conductive thin film for providing surface plasmons and a dielectric medium disposed at one side of the conductive thin film. A light source emits an incident light beam to the conductive thin film through the dielectric medium. A dielectric thin film having a surface to which a sample is attached is deposited at the surface of the conductive thin film opposite to the dielectric medium. The dielectric thin film is doped with active ions capable of fluorescing by being excited by the incident light beam. A photodetector receives and measures the intensity of fluorescence from the active ions to determine variations in refractive index for a sample. Quantification and qualification of the sample or measurement of the thickness of the sample (if the sample is a thin film) can be achieved from the refractive index variations.
Suitable photodetectors include a photodiode, a photomultiplier (PMT), a charge coupled device (CCD), and a photosensitive sheet. When the conductive thin film and the dielectric thin film are formed as arrays having a grid pattern, and a CCD or photosensitive sheet is used as the photoreceptor, an imaging system that images the sample with the contrast based upon fluorescence intensity variations between each array can be implemented.
In the AID WPR sensor and the imaging system using the same according to the present invention, the dielectric medium may be formed as a trapezoidal prism, and an optical filter or a lens may be further included. The trapezoidal prism is for directing the fluorescence in diverging directions toward the photodetector, and the optical filter enables the photodetector to receive pure light from the active ions by filtering out the wavelength of the incident light beam. The lens condenses the light from the active ions toward the photodetector.
Preferably, the active ions are derived from one selected from the group consisting of transition metal, rare-earth element, and organic dye. Preferably, the active ions have the ability to fluoresce by emitting light of a shorter wavelength than the incident light beam through two-photon or three-photon absorption. Suitable active ions include Tm3+ ions, Er3+ ions, Yb3+ ions, Ho3+xe2x80x94Yb3+ composite ions, Tm3+xe2x80x94Yb3+ composite ions, Er3+xe2x80x94Yb3+ composite ions, and Tm3+xe2x80x94Nd3+ composite ions. The wavelengths of the incident light beam and the light emitted from active ions are determined according to the type of active ions embedded in the dielectric thin film.
Preferably, the dielectric thin film is formed to be thick enough to produce a coupled plasmon-waveguide resonance mode and attenuated total reflection leaky mode coupled to surface plasma resonance, for example, to have a thickness of 100-700 nm.